魚 病 研 究 Fish Pathology,33(3),107-114,1998.9 Development of Thelohanellus hovorkai and Thelohanellus nikolskii (Myxosporea: Myxozoa) in Oligochaete Alternate Hosts Csaba Szekely1,*, Amina El-Mansy2, Kalman Molnar1 and Ferenc Baska1 1Veterinary Medical Research Institute, Hungarian Academy of Sciences, H-1581 Budapest, P.O. Box 18, Hungary 2National Institute of Oceanography and Fisheries, Cairo, Egypt (Received March 23, 1998) The development of Thelohanellus hovorkai, a myxosporean parasite of the connective tissues, and Thelohanellus nikolskii, a fin and scale parasite of common carp (Cyprinus carpio) was studied in experimentally infected oligochaetes Branchiura sowerbyi and Tubifex tubifex, respectively. After infection with mature spores of T. hovorkai, the development of actinosporean stages was first observed light microscopically in the gut of Branchiura sowerbyi 93 days after infection. Free actinospores of T. hovorkai were found in the lumen of the oligochaete's gut 101 days after infection. They were floating in water and showed a typical aurantiactinomyxon form. At 18-22•Ž, aurantiactinomyxon spores of T. hovorkai emerged from the worms 104 days after infec tion. The development of T. nikolskii was examined in Tubifex tubifex, from which aurantiactinomyxon spores were released 60 days after infection at 22-24•Ž. The diameter of spore body was 18.6ƒÊm and the lenght of caudal processes 29 gm for T. hovorkai, while 21.1 ƒÊm and 13.4 ƒÊm for T. nikolskii, respectively. The preva lence of aurantiactinomyxon infection in B. sowerbyi for T. hovorkai proved to be 16.7%, while in T. tubifex for T. nikolskii it was 12.5%. Key words: Thelohanellus hovorkai, Thelohanellus nikolskii, Myxozoa, aurantiactinomyxon, alternate host, Branchiura sowerbyi, Tubifex tubifex Since Wolf and Markiw (1984) published their species Shulman (1962) regarded only T. dogieli to be essential paper on the life cycle of Myxobolus cerebralis, valid, and considered T. nikolskii, T. amurensis, T. cyprini several other papers supporting their results have ap and T. hovorkai to be synonymous with T. dogieli, and peared. These papers (ElMatbouli et al., 1992; Kent T. acuminatus to be synonymous with T. fuhrmanni et al., 1993; Yokoyama et al., 1993; Uspenskaya, 1995) (Auerbach, 1909). The occurrence of a Thelohanellus revealed that in each case various Oligochaeta spp. species in Hungary was reported by Jeney (1979) who acted as alternate hosts in the development of various identified the parasites found on the fins of the common myxosporean species. carp as T. dogieli, and believed that it was introduced to To date, 7 Thelohanellus species have been described Europe by Far-Eastern carps. Molnar and Kovacs from the common carp. All of them were recorded Gayer (1981-82), who revitalized Akhmerov's classifi from Cyprinus carpio haematopterus in the Far-East. cation found two introduced species infecting fingerlings While studying parasites of the Amur River fishes, of the European common carp, namely T. nikolskii, a Akhmerov (1955, 1960) identified 5 host-and organ parasite of the fin and T. hovorkai, a parasite of the specific Thelohanellus species (T. acuminatus, T. mesenteries and conjunctive tissues. Moshu and amurensis, T. dogieli, T. nikolskii and T. hovorkai) from Molnar (1997) revealed that T. nikolskii can infect also the gills, liver, skin, fins and mesentery of the amur wild older specimens of the common carp. In older fish, carp, respectively. The number of Thelohanellus spe however, the plasmodia are located in the tips of the cies increased when Hoshina and Hosoda (1957), not scales. knowing Akhmerov's work (1955), described T. nikolskii The pathology of T. nikolskii was studied by Molnar under the name T. cyprini from the fins. Among these (1982) who reported large plasmodia growing on the finrays, destroying the structure of the cartilageous * E-mail: szekely@novell .vmri.hu elements and breaking down the fins. The pathology 108 Cs. Szekely, A. El-Mansy, K. Molnar and F. Baska of T. hovorkai was described by Yokoyama et al. (1998), All the worms were fed on some drops of granulated who found severe haemorrhages on the body surface of fish food, and pieces of chicken feces were added to in the affected common carp and designated the disease as crease the organic matter content of the mud. Normal "haemorrhagic thelohanellosis" . tap water was used throughout the experiments. The The intraoligochaete development of T. hovorkai has temperature of the room varied between 18 and 22•Ž already been demonstrated by Yokoyama (1997) who for T. hovorkai and between 22 and 24•Ž for T. nikolskii. found aurantiactinomyxon stages in experimentally in fected Branchiura sowerbyi. A comparative ultrasruc Laboratory experiment on T. hovorkai tural study on aurantiactinomyxon and raabeia stages has Two types of dishes were used. Some of the oli been carried out by Lom et al. (1997a). gochaetes were placed into small aquaria containing 5l During a research on the intraoligochaete develop of water, while others were transferred into small plas ment of myxosporeans the development of some tic cups of 0.5l volume. All dishes were permanently Myxobolus, Thelohanellus and Sphaerospora species aerated, and supplied regularly with fresh water to was studied experimentally under laboratory conditions. compensate for evaporation and to refresh the water. Within this program, successful experiments have been The aquaria contained about 100 Branchiura sowerbyi performed and some of their results have been described together with approximately 900 Tubifex tubifex and (El-Mansy and Molnar, 1997, 1998; El-Mansy et al., Limnodrilus hoffineisteri specimens, while the plastic a) or prepared for publication. The present paper1998 cups contained 50 Branchiura sowerbyi with about 40 reports on two experiments in which the oligochaete Tubifex and Limnodrilus specimens. The oligochaetes Branchiura sowerbyi was infected with myxospores of were infected by adding T. hovorkai spores to the dishes. T. hovorkai, and Tubifex tubifex was infected with An aquarium and a small cup containing the same spe myxospores of T. nikolskii, and in these worms actino cies and number of oligochaetes from the same stock sporean stages belonging to the aurantiactinomyxon were used as controls. type developed. The release of the actinosporean stages of T. hovorkai was followed by the methods described below: Materials and methods (1) Twice a week some of the oligochaetes were placed carefully under a coverslip and examined under Source of spores and oligochaetes microscope at 200•~ magnification for the presence of Spores of T. hovorkai were obtained from a single developmental stages. (2) From the third week after large plasmodium located in the gill arch of a one-year infection, oligochaetes of each species (B. sowerbyi, T. old common carp Cyprinus carpio, from a Hungarian tubifex and L. hoffineisteri) were placed into 2-ml cell fish farm, while spores of T. nikolskiiwere collected from well plates (Yokoyama et al., 1991) 3 times a week from several small plasmodia infecting the scales of four-year all the four dishes, and after a one-day incubation at old common carp from Kis-Balaton Water Reservoir. room temperature they were checked for the release of Branchiura sowerbyi (Beddard) specimens used in the actinospores under a microscope. (3) Every second experiments were collected from the mud of a fish pond, day, water from the aquaria and the plastic cups was selected from other oligochaetes, cleaned and placed in filtered through a fine mesh of 10 ƒÊm pore size. The 2-ml wells, and examined for the presence or absence of filtrates were taken up in a small volume of water and actinosporean infection (Yokoyama et al., 1991). The examined for the presence of actinosporean stages. (4) remaining Branchiura specimens were placed into an For histological examination the first oligochaetes were aerated aquarium and used in experiments when fixed only three months after infection. Within a month necessary. Only those Branchiura specimens were interval, 14 B. sowerbyi, 4 T. tubifex and 4 L. hoffineisteri used for the experiments which had been kept in labora specimens were fixed in Bouin' s solution, embedded in tory culture for at least 6 months. paraffin wax, cut into 5 ƒÊm thick sections, and stained Specimens of Tubifex tubifex and Limnodrilus with haematoxylin and eosin. One Branchiura speci hoffineisteri were collected from a muddy pool near the men releasing actinospores was fixed in 2% osmium top of a hill where no fishes live. They were transferred tetroxide, washed several times with cacodylate buffer, into sterilized mud and propagated in the laboratory in dehydrated and embedded in Durcupan ACM resin. aerated aquaria as parasite-free stocks. Oligochaetes Semithin sections (0.5-1.0 ƒÊm in thickness) were cut were identified according to Brinkurst (1963). with glass knives and stained with 0.1% methylene blue Extrapiscine development of two Thelohanellus 109 solution. 1 Laboratory experiment on T. nikolskii In this experiment only two plastic cups containing 20 Branchiura and approximately 50 Tubifex and Limnodrilus specimens each were used. One of the plastic cups contained oligochaetes exposed to myxo- spores of T. nikolskii, while the other cup served as con- trol. Only the water in these cups was examined after filtration using 10 p.m nylon mesh. Oligochaetes were squashed only after the first waterborne stages had been found. 2 Actinospores released from the oligochaetes were examined under a coverslip. They were recorded with the help of a video image program on videotapes (Szekely, 1997). Photos and drawings were made and measure- ments were recorded. All measurements in the descrip- tion are given as mean (range) in pm on the basis of 25 measured spores. The actinosporean stages of T. hovorkai and T. nikolskii were described according to the terminology of Janiszewska (1957) as modified by Lom et al. (1997b). Figs. 1-2. Waterborne aurantiactinospore of Thelohanellus hovorkai floating in the water 104 days post infection.